WO2012088146A2 - Biomarqueurs et leurs utilisations dans le pronostic et les stratégies de traitement du cancer du côlon droit et du cancer du côlon gauche - Google Patents

Biomarqueurs et leurs utilisations dans le pronostic et les stratégies de traitement du cancer du côlon droit et du cancer du côlon gauche Download PDF

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WO2012088146A2
WO2012088146A2 PCT/US2011/066233 US2011066233W WO2012088146A2 WO 2012088146 A2 WO2012088146 A2 WO 2012088146A2 US 2011066233 W US2011066233 W US 2011066233W WO 2012088146 A2 WO2012088146 A2 WO 2012088146A2
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colon cancer
patient
rcc
nox4
relapse
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WO2012088146A3 (fr
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Steven Buechler
Amanda B. HUMMON
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The University Of Notre Dame
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    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
    • C12Y106/03001—NAD(P)H oxidase (1.6.3.1), i.e. NOX1
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • Diagnostic tests for predicting relapse in colon cancer include the Oncotype DX test (Genomic Health). However, Genomic Health's test and others reports of a test for relapse in colon cancer is widely considered a failure.
  • the Oncotype DX Colon test identifies a small group of poor prognosis patients, but the test does not isolate good prognosis patients who can avoid further therapy, such as chemotherapy. Unfortunately, there does not exist a prognostic test for colon cancer that provides a consistent and accurate assessment of colon relapse risk in clinical practice.
  • RCC right-side colon cancer
  • LCC left-side colon cancer
  • the present invention provides powerful and highly significant biomarkers for quantifying risk of recurrence of location-specific colon cancer.
  • the present disclosure demonstrates that different processes dominate disease progression in left-side colon cancer (LCC) and right-side colon cancer (RCC), and that genes that are most predictive of relapse in LCC are much less significant in RCC, and vice-versa.
  • LCC left-side colon cancer
  • RCC right-side colon cancer
  • highly accurate and specific molecular tools are provided that can identify a patient as having LCC disease apart from those with RCC disease, and as a consequence of this, enable methods for highly accurate and effective techniques of prognosis assessment and treatment tailored to the disease type of the patient. In this way, methods for treating LCC and RCC as separate diseases are now possible.
  • the present disclosure identifies specific, previously unknown, colon cancer location specific biomarkers.
  • the specific colon cancer biomarkers are demonstrated to have a bimodal distribution pattern of expression.
  • Specific biomarkers for left side colon cancer (LCC) and for right side colon cancer (RCC) are provided.
  • LCC and RCC disease may be identified in the patient for example, by measuring expression levels of PRAC gene or by the clinical identification of the colon location site from which the colon cancer/tumor tissue sample was harvested. Then, the RCC and LCC biomarkers disclosed herein may be used to provide a predicted prognosis of the patient, from which a specific LCC or RCC clinical treatment plan may be formulated.
  • the specific and different genetic biomarkers of the invention separates each disease group population of colon cancer patients, the LCC disease group and RCC disease group population, into a good prognosis group and a poor prognosis group.
  • the LCC disease group population is divided into a poor prognosis LCC population group and a good prognosis LCC population group.
  • the RCC disease group population is divided into a poor prognosis RCC patient population group and a good prognosis RCC patient population group.
  • the biomarkers possess a bimodal distribution among these specific populations of colon cancer patients, and may be used as part of the presently described methods to provide location specific left-side or right-side colon cancer tumor disease assessment. Use of the biomarkers provides an improved and more accurate quantifier of risk of colon cancer relapse and of survival probability compared to tumor stage alone.
  • the bimodal genetic biomarkers for left-side colon disease and right side colon disease include NOX4, MMP3, CDX2 and FAM69A. These biomarkers have particular individual and distinct genetic expression level profiles. Differences in the expression level profiles and their distribution within a colon cancer population, RCC or LCC, correlates with the status of a patient as having a good prognosis of survival or as a patient with a bad prognosis of survival.
  • Genes NOX4 and MMP3 have a specific bimodal expression profile in left side colon cancer disease colon tissue that identifies a patient as having either a good or bad prognosis for 5-year relapse free survival.
  • Genes CDX2 and FAM69A have a specific bimodal expression profile in right side colon disease colon tissues that identifies a patient as having either a good or bad prognosis for 5-year relapse free survival.
  • LCC left side colon cancer
  • Such patients would be identified as in need of chemotherapy or other treatment to improve their chances of survival, whereas those expressing a low level of NOX4 are not at a higher risk for relapse, and therefore would not be in need of treatment such as chemotherapy or the like to improve their chances of a 5-year relapse free survival.
  • colon cancer disease in a population of colon cancer patients having right side colon cancer (RCC) disease, a patient whose tumor or tumor tissue (the tumor tissue being obtained from the right side of the colon in the patient) has a low gene expression level of a set of RCC-related genes comprising CDX2, FAM69A, or both, compared to a threshold expression value/level of a like-set of RCC related genes, is associated with poor prognosis and high expression levels with good prognosis.
  • low expression is relative to a threshold value/level of the like RCC-related genes, such as CDX2, in a population of RCC patients that have a known 5 -year history of relapse and relapse-free survival.
  • a method for prognosis of right-side colon cancer patients is provided.
  • colon cancer patients having right-side colon cancer disease with colon tissue expressing a low level of gene CDX2 compared to a threshold expression value/level for CDX2 are at higher risk for colon cancer relapse within a 5 -year post-surgical period.
  • This patient and/or patient population would be identified as in need of chemotherapy or other treatment to improve their chances of survival, whereas those expressing a high level of CDX2, are not at a higher risk for relapse, and therefore would not be in need of treatment such as chemotherapy or the like to improve their chances of a 5-year relapse free survival.
  • Relapse patients with RCC have been identified here to demonstrate accelerated cell cycle progression and elevated Wnt signaling.
  • Axin 2 is also identified to be downregulated in RCC relapse patients.
  • improved methods for managing the clinical care of a patient having been diagnosed with colon cancer are provided.
  • the present invention provides, in some aspects, a method for identifying the best clinical management for the treatment of a left-side colon cancer (LCC) patient, following surgical intervention to remove the cancer, as well as a method for identifying the best clinical management for the treatment of a right side colon cancer (RCC) patient following surgical removal of the cancer.
  • LCC left-side colon cancer
  • RCC right side colon cancer
  • the assessment of gene expression levels of a defined panel of genes may be measured using GeneChip®. or microarray technology. While any number of standard microarray platforms known to those of skill in the art may be used, an example of one commercially available microarray is the GeneChip®. (Affymetrix®.).
  • Other RCC indicative molecules associated with relevant RCC biological pathways include cyclic dependent kinase inhibitor 2B (CDKN2B), growth arrest and DNA damage inducible, alpha (GADD45A) and cyclin Dl (CCND1).
  • a significant percentage of RCC patients that experience a colon cancer relapse after surgical intervention have been determined, according to the methods of the present invention, to present right side colon cancer samples that demonstrate low expression levels of caudal type homeobox 2(CDX2).
  • low expression is relative to the DCX2 expression levels of the RCC cancer tumors in a population of all RCC cancer patients.
  • the present model provides a highly prognostic indicator concerning relative risk for recurrent colon cancer relapse.
  • Prior description of the use of the CDX2 gene as a colon cancer has been mixed, with the CDX2 gene having been used only in identifying a patient as having a cancer of the right side of the colon or not. 23 While some report CDX2 as increased in colon tumors, others report it decreased.
  • a RCC patient with low CDX2 expression levels accordingly, would likely be proscribed a more aggressive, post colon surgery, treatment regimen, such as chemotherapy and/or radiation therapy.
  • a RCC patient with relatively high expression levels of CDX2 has a lower risk of recurrence than the overall population of patients diagnosed with RCC disease.
  • the 5-year expected survival probability is sufficiently high that the patient would not benefit from systemic chemotherapy, radiation therapy, or other post-colon cancer resection surgery.
  • Table 1 provides a chart of the biomarker genes, and the probes employed to assess the gene expression. These commercially available gene probe families are provided here for example only, as other genetic probes for the identified biomarker genes may be devised by one of skill in the at and employed in the practice of the present invention employing the teachings of the present disclosure.
  • the good prognosis component of CDX2 in the right-side samples in GSE14333 contains 84% of the samples.
  • the good prognosis component defined by using both CDX2 and FAM69A contains 80% of the samples.
  • NOX4 is largely unexpressed in RCC. This means that a test that examines N0X4 in a patient having RCC disease will result in a false "good prognosis" assessment of the patient.
  • RCC refers to a tumor tissue and/or cancerous tissue that is identified from tissue harvested from the right side of the colon.
  • the right side of the colon will be understood in the description of the present invention as that part of the human colon that extends from the cecum or ascending colon and extends through the transverse colon, excluding the appendix.
  • NOX4 has been implicated in cancer development by reactive oxygen species (ROS) in several forms of cancer 12 , but NOX4 has not been previously implicated in colon cancer progression.
  • ROS reactive oxygen species
  • a higher percentage of LCC patients that experience a colon cancer relapse after surgical intervention have been determined, according to the methods of the present invention, to present left side colon cancer samples with a higher expression levels of NOX4. These patient samples also evidence elevated integrin-binding sialoprotein (IBSP), and lower expression levels of matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3).
  • IBSP integrin-binding sialoprotein
  • MMP3 matrix metallopeptidase 3
  • MMP3 matrix metallopeptidase 3
  • a higher NOX4 expression level in a left-side colon cancer tissue would be indicative of a higher risk of colon cancer relapse.
  • this patient population would more likely benefit in a higher probability of increased survival without relapse and decreased risk of colon cancer metastasis if additional, post-colon surgery, treatments were administered, such as chemotherapy and/or radiation therapy.
  • a LCC patient with low NOX4 expression levels has a high probability of relapse-free survival for 5 years. Such a patient is unlikely to benefit from systemic chemotherapy, radiation therapy, or other post colon cancer resection surgery procedure.
  • a panel of gene biomarkers for good prognosis LCC patients may be obtained by combining 2 or more genes in Table 2.
  • a set of good prognosis patients is the intersection of the good prognosis components of the individual genes in the panel.
  • Table 2 provides a chart of the biomarker genes, and the probe employed to assess the gene expression.
  • a panel of gene biomarkers for good prognosis LCC patients is obtained by combining 2 or more genes in Table 5.
  • a set of good prognosis patients is the intersection of the good prognosis components of the individual genes in the panel.
  • a LCC patient with high NOX4 would likely be proscribed an aggressive post colon surgery treatment regimen. It is anticipated that this population of patients would benefit from an increase in probability of relapse free survival, or decreased probability of colon cancer metastasis, with subsequent aggressive clinical treatment, such as chemotherapy and/or radiation therapy.
  • the good prognosis component of NOX4 in the left-side samples in GSE14333 contains 56% of the samples.
  • the good prognosis component defined by using both NOX4 and MMP3 contains 51% of the samples.
  • LCC refers to a tumor tissue and/or cancerous tissue that is identified from tissue harvested from the left side of the colon.
  • the left side of the colon will be understood in the description of the present invention as that part of the human colon that begins at the left splenic flexure, includes the descending colon and ends with the sigmoid, but does not include the rectum.
  • NADPH oxidase 4 (as compared to a threshold expression value/level of the gene in a LCC population of patients that have at least a 5 -year history of relapse or as relapse-free) is highly predictive of relapse in post-colon surgery patients.
  • CDX2 has normal expression levels in most LCC relapse cases.
  • the present invention also provides a panel of genetic probes for assessing 5 year survival probability without relapse in a patient population having a cancerous leftside colon (LCC) tumor. These genetic probes are described herein in Tables 2 and 4.
  • the present invention also provides a panel of genetic probes for assessing 5 year survival probability without relapse in a patient population having a cancerous right- side colon (RCC) tumor. These genetic probes are described herein at Tables 1 and 5.
  • Methods of the invention can be utilized in a number of different applications.
  • diagnostic chips can be fabricated based on the identification of the diagnostic genes, such as the ones identified herein at Tables 4 and 5. Such chips would be useful in clinical settings, as it would allow clinicians to diagnose a particular type of colon cancer from a relatively small set of genes, instead of purchasing entire gene sets.
  • the methods of the present invention may take the form of a diagnostic and/or screening tool that is provided in the form of an array of genetic probes specific for the colon cancer biomarkers described herein.
  • array refers to a grouping or an arrangement, without being necessarily a regular arrangement.
  • An array comprises preferably at least 2, more preferably 5 different sets of detection molecules or patient samples.
  • the array of the present invention comprises at least 50 sets of detection molecules or patient samples, further preferred at least 100 sets of detection molecules or patient samples.
  • the detection molecule can be for example a nucleic acid probe, such as the nucleic acid probes provided at Table 4 (for LCC disease), Table 5 (for RCC disease), or in some embodiments, the nucleic acid probes of both Tables 4 and 5.
  • the described array can be used in a test system according to the invention.
  • the array can be either a micro array or a macro array.
  • the detection molecules are immobilized to a solid surface or support or solid support surface. This array or microarray is then screened by hybridizing nucleic acid probes prepared from patient samples or by contacting the array with proteinaceous probes prepared from patient samples.
  • the support can be a polymeric material such as nylon or plastic or an inorganic material such as silicon, for example a silicon wafer, or ceramic.
  • glass Si02
  • the glass can be a glass slide or glass chip.
  • the glass substrate has an atomically flat surface.
  • Methods of the invention can also be used for identifying pharmaceutical targets. Pharmaceutical companies can utilize methods of the invention to determine which genes to target in efforts to target specific right-side colon disease or left-side colon disease.
  • the method may further include the step of producing a report indicating a RCC or LCC prognosis for the colon cancer patient based on the expression levels and a comparison to other patients with similar expression levels, and optionally, calculating a recurrence score based on the expression levels.
  • any of the steps of the methods may be performed by a computer.
  • the expression level of the gene panel is performed by microarray analysis with multi-state probes specific to the genes of the gene panel.
  • a computer running a software program analyzes gene expression level data from a patient, compares that data to a distribution of expression levels from a population of colon patients having a RCC or LCC disease state, and determines whether the patient's expression levels have a +/- status for each gene identified herein as informative to an RCC or LCC prognosis, respectively.
  • the +/-status of a LCC or RCC patient's colon tumor tissue gene expression is determined based on comparing that patient's colon sample tissue level of gene expression to the density distribution of gene expression from all LCC or RCC patients in a sample group.
  • density distribution of expression levels from the sample population is determined based on mixture model fit statistical method which is a statistical method know to those of skill in the art.
  • a key discovery according to one aspect of the invention as described herein is that the expression by LCC or RCC cancer patients of multi-state genes, as described herein, presents at least a bimodal distribution when the expression level density distribution is determined using the mixture model fit method.
  • the computer software is capable of determining the prognosis for the patient as being good or poor.
  • the software is capable of generating a report summarizing the patient's gene expression levels and/or the patient's (+) or (-) status scores, and/or a prediction of the likelihood of long term survival of the patient and/or the likelihood of recurrence or metastasis of the patient's LCC or RCC disease condition.
  • the computer program is capable of performing any statistical analysis of the patient's data or a population of patient's data as described herein in order to generate the + or - status of the patient.
  • the computer program is also capable of normalizing the patient's gene expression levels in view of a standard or control prior to comparison of the patient's gene expression levels to those of the patient population.
  • the computer is capable of ascertaining raw data of a patient's expression values from, for example, immunohistochemical staining or a microarray, or, in another embodiment, the raw data is input into the computer.
  • ROS Reactive Oxidative Species
  • ROS reactive oxidative species
  • Interference RNA for NOX4 may be used to inhibit the aggressiveness of LCC tumors by reducing ROS production.
  • ROS production in a colon cancer cell line, SW620 was reduced by inhibiting NOX4 mRNA using interference RNA.
  • ROS production in LCC may also be inhibited by interfering with the activity of the NOX4 protein using an antibody.
  • the methods of the present invention are carried out with colon sample material such as a colon tumor tissue sample which already has been isolated from the human body. Subsequently the sample material can be fractionated and/or purified. It is for example possible, to store the sample material to be tested in a freezer and to carry out the methods of the present invention at an appropriate point in time after thawing the respective sample material.
  • colon sample material such as a colon tumor tissue sample which already has been isolated from the human body.
  • the sample material can be fractionated and/or purified. It is for example possible, to store the sample material to be tested in a freezer and to carry out the methods of the present invention at an appropriate point in time after thawing the respective sample material.
  • the pathological condition of the afflicted individual can be further exacerbated by formation of metastasis.
  • the present invention may be used to discriminate and identify early colon cancer, thus permitting the detection of the colon cancer disease at an early and still benign stage, an early stage or benign stage and/or early colon tumor stages. The early detection enables the physician to timely remove the colorectal adenoma and to dramatically increase the chance of the individual to survive.
  • the expression levels from the population of right side colon cancer (RCC) disease patients or left side colon cancer (LCC) disease patients for each gene in the colon cancer gene panel comprises a bimodal density distribution such that a statistically significant threshold exists between the two modes, whereby expression levels on one side of the threshold are deemed high and expression levels on the other side of the threshold are deemed low.
  • the LCC or RCC patient sample is classified as demonstrating a relatively low expression level or a relatively high expression level of the informative gene or set of genes for LCC or RCC as defined here (See Tables 1 and 2), and the expression level is compared to a threshold expression value/level of a like gene or set of like genes.
  • the prognosis in the RCC or LCC patient is then assessed based upon the specific gene expression data obtained from an existing pool of genetic expression profile data collected from the RCC or LCC disease patients, respectively, having a known positive 5 year colon cancer free survival history and a specific LCC or RCC genetic profile expression level data set.
  • this data set is a data set of mRNA expression values for NOX4 and MMP3 (for LCC), and CDX2 and FAM69A (for RCC).
  • the expression level profiles and diagnostic methods of the present RCC and LCC disease models provided here employing the bimodal genes identified for RCC and LCC are completely independent of and unrelated to the estrogen receptor (+) or (-) status of the tissue sample and any bimodal gene identified for breast cancer, and is unrelated to assessment of breast cancer prognosis or risk of relapse for breast cancer.
  • the density distribution is determined by mixture model fit statistical analysis.
  • the expression levels of each RCC or LCC gene from the respective population of RCC or LCC patients forms a density distribution of at least two or more modes and a statistically significant threshold exists between the two or more modes. Expression levels on one side of a defined threshold are deemed positively correlated with mortality and expression levels on the other side of a defined threshold are positively correlated with survival.
  • the density distribution is determined by mixture model fit statistical analysis.
  • a data set of mRNA expression values may be generated using, for example, an Affymetrix. microarray. One array may be generated for each patient in the cohort. Consider an array probe p such that increased expression is statistically significant in a univariate Cox proportional hazard model of relapse.
  • p is designated multi-state in this cohort if the density distribution can be partitioned into two components: a large normal component of expression values below a threshold c, and a long right tail with expression values above c.
  • FIG. 1 Elevated NOX4 expression is a significant predictor of relapse in leftside colon cancer
  • (a) The density distribution of NOX4 expression in the left-side tumors on GSE14333 with Dukes Stage A, B or C shows a large component with low baseline expression and a tail of elevated expression values. Individual expression values are indicated with hatch marks at the lower edge.
  • the multistate methodology divides the samples at an expression value of 3.1; the samples with expression below 3.1 are in the NOX4- component, and those with expression values above 3.1 are in NOX4+.
  • the relapse event vector gives a sample the value 0 if it is relapse-free for 60 months and the value 1 otherwise.
  • the boxplot of NOX4 expression versus the relapse event vector illustrates the significance of the dependence
  • (c) The Kaplan-Meier curves for the NOX4- and NOX4+ components plot the expected survival probabilities for the components in the left-side tumors.
  • the 5-year expected survival probability for NOX4- is 0.89 95%CI (0.80 - 0.99) and for NOX4+ it is 0.51 95%CI (0.37 - 0.70).
  • a Cox proportional hazard model whose only variable is an indicator for the NOX4 components has a logrank test p-value 1.2 x 10 "4 .
  • NOX4- contains 53 samples and NOX4+ contains 42 samples
  • (d) The corresponding Kaplan-Meier plots for the NOX4 components in the right-side tumors shows a distinctly lower connection with relapse than on the left side.
  • the 5-year expected survival for NOX4- on the right side is 0.82 95C1% (0.73 - 0.93) and for NOX4+ it is 0.73 95%CI (0.56 - 0.95).
  • On the right side only 28 samples are in NOX4+ and 72 are in NOX4-.
  • Low CDX2 expression is a significant predictor of relapse in right- side colon cancer
  • (a) The density distribution of CDX2 expression in the right-side tumors on GSE14333 with Dukes Stage A, B or C follows a bimodal distribution.
  • the multistate methodology divides the samples at an expression value of 4.76; the samples with expression below 4.76 are in the CDX2- component, and those with expression values above 4.76 are in CDX2+.
  • (b) The boxplot of CDX2 expression versus the relapse event vector illustrates the significance of the dependence. In this case, low CDX2 expression is predictive of relapse
  • (c) The Kaplan-Meier curves for the CDX2+ and CDX2- components plot the expected survival probabilities for the components in the right-side tumors.
  • the 5-year expected survival probability for CDX2+ is 0.88 95%CI (0.80 - 0.96) and for CDX2- it is 0.39 95%CI (0.15 - 0.78).
  • a Cox proportional hazard model whose only variable is an indicator for the CDX2 components has a logrank test p- value 1.68 x 10 "7 .
  • CDX2+ contains 86 samples and CDX2- contains 16 samples,
  • the corresponding Kaplan-Meier plots for the CDX2 components in the left-side tumors shows a distinctly lower connection with relapse than on the right side.
  • the 5-year expected survival for CDX2+ on the left side is 0.75 95C1% (0.66 - 0.86) and for CDX2- it is 0.35 95%CI (0.12 - 1.0).
  • Figure 3 Expression of endogenous NOX4 in human colon cancer cell lines. RTPCR was performed using RNA from several human colon cancer cell lines (HCT116, HT29, SW480 and SW620) with specific primers for NOX4. NOX4 mRNA levels were quantified using the comparative CT method relative to levels of hypoxanthine phosphoribosyltransfease (HPRT). The fold change in expression (Log 10 ) was normalized to normal colon NOX4 mRNA levels. Three experiments were conducted in triplicate. Bars represent the median fold change (Log 10 ) and the error bars represent the standard deviation.
  • FIG. 5 Targeted NOX4 knockdown decreases superoxide production in SW620 cells.
  • SW620 cells were transfected with NOX4 siRNA and assayed for superoxide production by the chemiluminescent method.
  • Superoxide producing activity of AllStar negative siRNA transfected cells are set as 100%.
  • Each bar represents the mean data from 2 independent transfections, with error bars representing the S.D. for percentage of activity.
  • Figure 6 Flow chart of screening a colon cancer patient. This flow chart may be embodied as a software program that may be used in an automated clinical tool for screening patient samples.
  • the present invention in a general and overall sense, provides for biomarkers specific for left side colon cancer (LCC) and for right side colon cancer (RCC), as well as the use of these markers in providing powerful diagnostic and prognostic tools for predicting survival probabilities of patients with each disease.
  • LCC left side colon cancer
  • RCC right side colon cancer
  • the invention provides for a method of measuring expression levels of the biomarker NOX4, MMP3, or a combination of these as an assessment indicator of left-side colon cancer (LCC) prognosis.
  • LCC left-side colon cancer
  • the invention provides for a method of measuring expression levels of the biomarker CDX2, FAM69A, or a combination of these, as an assessment indicator of right-side colon cancer (RCC) prognosis.
  • RCC right-side colon cancer
  • a clinically applicable version of the present methods may use RT-PCR to measure mRNA obtained from formalin-fixed, paraffin-embedded (FFPE) colon tissue.
  • FFPE paraffin-embedded
  • the present invention demonstrates that different processes dominate progression to relapse in LCC and RCC.
  • genes that are most predictive of relapse in LCC are much less significant in RCC, and vice-versa.
  • elevated expression of NADPH oxidase 4 (NOX4) is highly predictive of relapse, while NOX4 is largely unexpressed in RCC.
  • NOX4 is largely unexpressed in RCC.
  • the NOX family of genes has been implicated in cancer development by reactive oxidative species (ROS) in several forms of cancer 14 , but NOX4 has not been previously implicated in colon cancer progression.
  • ROS reactive oxidative species
  • CDX2 caudal type homeobox 2
  • NOX4 The role of NOX4 in colon cancer is further investigated using the SW620 lymph-node metastasis colon adenocarcinoma cell line and RNA interference. NOX4 is expressed in the SW620 cell line, and application of NOX4 siRNA causes a significant reduction in ROS production.
  • sample material is also designated as “sample”.
  • biomarker is meant to designate a protein or protein fragment or a nucleic acid which is indicative for the incidence of the colorectal adenoma and/or colorectal carcinoma. That means the “biomarker” is used as a mean for detecting colorectal adenoma and/or colorectal carcinoma.
  • a "multi-state gene” is defined as a gene capable of differential levels of expression within a LCC or RCC disease patient population such that the expression levels of the gene in the LCC or RCC disease patient population permits the patient population to be divided into at least two or more distribution groups based on density distribution according to statistical analysis of the expression level of specific LCC- associated (such as NOX4 and MMP3) or RCC associated (such as CDX2 and FAM69A) informative genes. For example, in one embodiment, the expression levels are divided into two groups based on a mixture model fit of expression levels of the gene of interest.
  • the gene is a multi-state gene.
  • a gene is multi- state if the density distribution of gene expression for a particular gene of interest is partitioned into at least two components, a large normal component of expression values above a threshold c, and a long left tail with expression values below c.
  • the fitting algorithm actually produces, for each point and component, a posterior probability that the point is in that component.
  • the point is assigned to the component whose associated posterior probability is maximal.
  • ⁇ For a point p that is well- classified in, say, component 1, the posterior probability that p is in C2 will be very small.
  • Ratios are .ltoreq.l, with numbers close to 1 representing well-isolated components. Ratios are used to measure the ability of a mixture model fit to describe distinct states.
  • the components defined by a fit of a pair of gaussian distributions consist of a pair of unbroken intervals. That is, there is a cutoff c so that one component consists of the values ⁇ c and the other component the values greater than or equal to c. In this way, mixture models can be used to calculate a threshold for dividing a vector into high and low components.
  • a standard measure of the quality of a mixture model fit is the likelihood, which is the product, over all points, of the maximal posterior probabilities.
  • the likelihood can be used to decide, for example, if a fit with a pair of gaussian distributions is better than a fit with a single gaussian, or if a fit with Gamma distributions is better than a fit with gaussian distributions.
  • Even better measures are AIC and BIC which adjust likelihood by the degrees of freedom. These measures play a part in defining the notion of a multi-state probe.
  • mixture models were fit using the flexmix R package (Leisch, 2004).
  • Probe means a polynucleotide molecule capable of hybridizing to a target polynucleotide molecule.
  • the probe could be DNA, cDNA, RNA, or mRNA.
  • a probe is fixed, for example, by a covalent bond, to a solid state apparatus such as a microarray.
  • the probe and the target may hybridize, for example, under stringent, or moderately stringent conditions.
  • a probe may be labeled, for example, with a fluorescent or radiolabel to permit identification.
  • a probe is of a sufficient number of base pairs such that it has the requisite identity to bind uniquely with the target and not with other polynucleotide sequences such that the binding between the target and the probe provides a statistically significant level of accurate identification of the target molecule.
  • a probe's ability to bind a target is correlated to a statically significant prognostic indicator of a defined disease state as determinable using an identified panel of genes of interest.
  • the target is mRNA and the probe is a complementary piece of DNA or cDNA.
  • the target is cDNA or DNA and the probe is a complementary piece of mRNA.
  • the target is cDNA or DNA and the probe is a complementary piece of DNA.
  • multi-state probe is meant, in one embodiment, as a probe capable of hybridizing with a target polynucleotide molecule encoding a LCC or RCC specific multi-state gene.
  • a "multi-state LCC or RCC probe” means a probe capable of hybridizing with a target polynucleotide molecule encoding a relevant portion or fragment of a LCC or RRC multi-state gene, respectively.
  • the target polynucleotide molecule may be mRNA.
  • a LCC or RCC multi-state probe (see Tables 1, 2, 4 or 5, respectively) is fixed to a solid state apparatus such as a microarray by, for example, a covalent bond.
  • hybridization between the probe and the target occurs under stringent conditions.
  • hybridize or “hybridizing” or “hybridization” refers to the formation of double stranded nucleic acid molecule between complementary sequences by way of Watson-Crick base-pairing. Hybridization can occur at various levels of stringency according to the invention. "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature.
  • "Stringent conditions” or “high stringency conditions”, as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 inM sodium chloride, 75 mM sodium citrate at 42° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate
  • Modely stringent conditions may be identified as described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and % SDS
  • An example of moderately stringent conditions is overnight incubation at 37° C.
  • microarray refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
  • differentially expressed gene refers to a gene whose expression is activated to a higher or lower level in a subject suffering from a LCC or RCC disease, relative to its expression in a normal or control subject.
  • the terms also include genes whose expression is activated to a higher or lower level at different stages of the same disease. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.
  • Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically cancer, or between various stages of the same disease.
  • Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
  • differentiated gene expression is considered to be present when there is at least an about two-fold, preferably at least about four-fold, more preferably at least about sixfold, most preferably at least about ten-fold difference between the expression of a given gene in normal and diseased subjects, or between various stages of disease development in a diseased subject.
  • RNA transcript is used to refer to the level of the transcript determined by normalization to the level of reference mRNAs, which might be all measured transcripts in the specimen or a particular reference set of mRNAs.
  • prognosis is used herein to refer to the prediction of the likelihood of LCC or RCC cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as RCC or LCC disease.
  • prediction is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary LCC or RCC tumor and/or chemotherapy for a certain period of time without cancer recurrence.
  • the predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities is likely.
  • a treatment regimen such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy
  • long-term survival is used herein to refer to survival for at least 3 years according to one embodiment, at least 8 years according to a more preferred embodiment, and at least 10 years according to a most preferred embodiment, following surgery or other treatment.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • the "pathology" of cancer includes all phenomena that compromise the well- being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • node negative cancer such as “node negative” colon cancer, is used herein to refer to cancer that has not spread to the lymph nodes.
  • gcrma refers to a method know to those of skill in the art whereby raw data obtained from an Affymetrix.® microarray is normalized.
  • Normalization refers to statistical normalization.
  • a normalization algorithm is the process that translates the raw data for a set of microarrays into measure of concentration in each sample.
  • a survey of methods for normalization is found in Gentleman, et al.
  • a microarray chip assesses the amount of mRNA in a sample for each of tens of thousands of genes. The total amount of mRNA depends both on how large the sample is and how aggressively the gene is being expressed. To compare the relative aggressiveness of a gene across multiple samples requires establishing a common baseline across the samples. Normalization allows one, for example, to measure concentrations of mRNA rather than merely raw amounts of mRNA.
  • Bioly homogeneous refers to the distribution of an identifiable protein, nucleic acid, gene or genes, the expression product(s) of those genes, or any other biologically informative molecule such as a nucleic acid (DNA, RNA, mRNA, iRNA, cDNA, etc.), protein, metabolic byproduct, enzyme, mineral, etc., of interest that provides a statically significant identifiable population or populations that maybe correlated with an identifiable disease state of interest.
  • Low expression or “low expression level(s),” “relatively low expression,” or “lower expression level(s)” and synonyms thereof, according to one embodiment of the invention, refers to expression levels, that based on a mixture model fit of density distribution of expression levels for a particular multi-state gene of interest falls below a threshold c, whereas "high expression,” “relatively high,” “high expression level(s)” or “higher expression level(s)” refers to expression levels failing above a threshold c in the density distribution.
  • the threshold c is the value that separates the two components or modes of the mixture model fit.
  • the term "healthy individual” or “healthy individuals” is meant to designate individual(s) not diseased of colorectal adenoma and/or colorectal carcinoma. That is to say, the term “healthy individual(s)” is used only in respect of the pathological condition of colorectal adenoma and/or colorectal carcinoma and does not exclude the individual to suffer from diseases other than colorectal adenoma and/or colorectal carcinoma.
  • the term "derivative thereof is meant to describe any modification on DNA, mRNA or protein level comprising, e.g., the truncated gene, fragments of said gene, a mutated gene, or modified gene.
  • the term "gene” includes nucleic acid sequences, such as DNA, RNA, mRNA or protein sequences or oligopeptide sequences or peptide sequences.
  • the derivative can be a modification which is an result of a deletion, substitution or insertion of the gene.
  • the gene modification can be a result of the naturally occurring gene variability.
  • naturally occurring gene variability means modifications which are not a result of genetic engineering.
  • the gene modification can be a result of the processing of the gene or gene product within the body and/or a degradation product.
  • the modification on protein level can be due to enzymatic or chemical modification within the body.
  • the modification can be a glycosylation or phosphorylation or farnesylation.
  • the derivative codes for or comprises at least 5 amino acids, more preferably 10 amino acids, most preferably 20 amino acids of the unmodified protein.
  • the derivative codes for at least one epitope of the respective protein.
  • the term "patient” as used in the present application covers humans as well as non-human beings such as animals.
  • the animals are preferably selected from the group consisting of rodents, e.g., mouse, rat, hamster, and other animals, e.g., guinea-pig, rabbit, hare, dog and pig.
  • These animals can be used to specifically induce certain disease states, like colorectal adenoma and colorectal carcinoma, for research purposes.
  • the induction of said disease states can, for example, be effected by treatment of the animals, for example, with radioactive or chemical substances known to induce colorectal cancer or colorectal adenoma disease state.
  • the disease states can also be induced using viral transfection systems. It is also possible to use genetically modified animals, in which one or more specific gene function(s) has/have been altered, or knock-out animals such as knock-out mice in which a specific gene function has been deleted.
  • the term "compound” can be one or more chemical substances, an antibody, protein, peptide, antisense mRNA, small molecular drug, or combinations thereof.
  • the compound can also be replaced by irradiation, e.g., X-ray, or combinations of compounds and radiation can be used.
  • a good prognosis may be defined as a prognosis in which a patient is determined to be unlikely to benefit from cancer treatment such as chemotherapy or radiation, for example, subsequent to a colon cancer surgical procedure. This may be the case where the expression level of the identified bimodal gene or combination of genes for LCC or RCC disease is negatively correlated with mortality.
  • a poor prognosis patient is used to define a patient that is likely to benefit from further cancer treatment such as chemotherapy or radiation, for example, subsequent to a colon cancer surgical procedure. This may be the case where the expression level of the identified bimodal gene or combination of genes for LCC or RCC disease is positively correlated with mortality.
  • Example 1 Identification of RT-PCR primer-probes that measure in FFPE tissue the m RN A species targeted by the ap-Colon microarray probes
  • mRNA will be extracted from a number of colon cancer cell lines as well as from paraffin (FFPE) blocks prepared from these cell lines. This will enable direct assessment of the probes in the FFPE material and comparison with the "fresh state”. Initial assessment will be performed using 13 different assay primer-probes pairs (8 from ap-Colon (two per gene) and 5 normalization controls). All assays will be performed in triplicate. The probes will be verified as providing comparable results in fresh tissues (cell lines) and matched FFPE counterparts. Quantitative RT-PCR with AACT methods for data analysis will be used to assess the utility of the probes. If suitable primer-probes cannot be found for the initial choice of genes, the list will be screened to identify replacement genes found in the development of ap-Colon. The RL-COLON pair of tests will use the primer-probes identified here.
  • RL-COLON Use RL-COLON to verify differential expression of each gene in the panel in archival colon cancer tissue with varying stages.
  • the tissue obtained will be divided in training and validation sets.
  • the training set will be used to find thresholds between high and low expression levels of the genes in RL-COLON, replacing the thresholds in the microarray-based ap-Colon.
  • the validation set is used to verify that RL-COLON is sufficiently predictive and prognostic to guide treatment decisions.
  • the present example is provided to present the various materials, methods and statistical tools employed in the development and practice of the present invention.
  • KEGG pathway analysis The Kyoto Encyclopedia of Genes and Genomes (KEGG) http://www.genome.ip/kegg/kegg2.html identifies the component genes in selected pathways.
  • the BioConductor package hgul33plus2 . db is used to associate array probes with pathways.
  • Multistate survival models In Buechler, et al. 2 , a method of defining survival models based on gene expression data is presented.
  • an array probe (gene) is called multistate if the probe's expression values naturally divide samples into two distinct subtypes, much like the bimodality of the ESR1 gene divides samples into ER+ and ER- subgroups.
  • p there is a threshold c such that the samples with expression values above c, denoted p+, form one component, and the samples with expression values below c, denoted p-, form the second component.
  • multistate probes that arise in survival models in cancer, one of the components is approximately normally distributed with a narrow variance, and the other smaller component is a tail to the right or left. Many genes have nearly normal expression distributions, hence are not considered multistate.
  • the precise definition of a multistate probe is given in Buechler, et al. 2 [0122] Colorectal cancer often develops through a specific genetic progression .
  • the expression vector for a multistate probe is replaced by a binary variable which is 0 in the component of good prognosis samples and 1 in the poor prognosis component.
  • the significance of a multistate probe in a survival model is measured by the p-value of a logrank score of a Cox proportional Hazard Model (abbreviated CPH) using only the probe's binary variable.
  • CPH Cox proportional Hazard Model
  • Colorectal cancer cell lines HCT-116, HT29, SW480, SW620 and SW837 were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and were maintained in RPMI 1640 medium (Invitrogen, Gaithersburg, MD) containing 10% fetal bovine serum (Thermo Scientific, Pittsburgh, PA) and 2mM L- glutamine (Invitrogen, Gaithersburg, MD) and grown in 5% C0 2 at 37°C.
  • siRNA NOX4_5 and siRNA NOX4_8 correspond with the following sequences:
  • siRNAs used were obtained from Oiagen Inc. (Germantown, MD).
  • Oiagen Inc. Germantown, MD
  • each siRNA (2 pmol) was added to individual wells in a 96-well plate in 25 ⁇ , of serum-free RPMI and complexed with 0.5 ⁇ , Oligofectamine transfection reagent (Invitrogen, Gaithersburg, MD) in 25 ⁇ _ of serum-free RPMI. The resulting mixture was allowed to complex for 30 min at ambient temperature.
  • SW620 cells (5000 cells/well) were added in 50 pL RPMI supplemented with 20% FBS to yield transfection mixtures consisting of 20 nM siRNA in RPMI with 10% FBS.
  • the final mixture was incubated at ambient temperature for 45 min before being placed in an incubator in 5% C0 2 at 37°C.
  • transfections were performed as described above except that they were conducted in 6-well plates and all reagent amounts were scaled up 30-fold.
  • each siRNA was evaluated by comparing cell viability, mRNA or ROS levels with those found in cells transfected with negative-control siRNA. siRNA knockdown was validated using real-time PCR.
  • Cell viability assay As a surrogate marker for cell viability, the reduction of resazurin to resorufin was measured in transfected cells using the Cell Titer-Blue Cell Viability assay (Promega, Madison, WI). Triplicate transfections for each siRNA duplex were set up in a 96-well plate at a concentration of 5,000 cells/well. After 72 and 96 hr, 20 ⁇ , of CellTiter-Blue reagent was placed in each well and incubated for 1 hr.
  • Reduction of resazurin to resorufin, read as fluorescence emission (560Ex/590Em) was measured using a plate reader (Spectramax M5, Molecular Devices, Sunnyvale, CA). Viability of transfected cells was compared with cells transfected with the negative control siRNA. Additional cells transfected with Qiagen' s All Star Death control were used as a positive control.
  • RNeasy Mini kit Qiagen, Germantown, MD
  • Normal human colon RNA isolated postmortem from a donor was purchased from Ambion (Applied Biosystems, Foster City, CA). Nucleic acid quantity, quality and purity were determined using a Nanodrop 2000 UV-VIS spectrophotometer (Nanodrop, Rockland, DE).
  • cDNA was generated using the High-Capacity Reverse Transcriptase cDNA kit (Applied Biosystems, Foster City, CA) and 1.0 ⁇ g of total RNA according to the manufacturer's instructions. Quantitative PCR reactions were performed using the following primer sequences (Operon, Huntsville, AL): hypoxanthine phosphoribosyltransferase 1 (HPRT1), HPRT1 For
  • Quantitative PCR was performed with a real-time PCR system, StepOnePlus (Applied Biosystems, Foster City, CA). Reactions were conducted with 300 ng of cDNA, in a final volume of 25 ⁇ ,.
  • the PCR mixture contained SYBR Green (Applied Biosystems, Foster City, CA) and 0.6 nmol of each primer (forward and reverse).
  • the levels of transcripts were quantified using the comparative CT method relative to levels of hypoxanthine phosphoribosyltransfease (HPRT1). All samples were analyzed in triplicate wells with the median of each measurement used for CT calculations.
  • SW620 cells were assayed for superoxide production 48 hr post transfection with siRNA NOX4_5, siRNA NOX4_8 or negative control siRNA.
  • Transfected cells were washed with phosphate-buffered saline (Invitrogen, Gaithersburg, MD) and collected into a centrifuge tube.
  • Superoxide production was measured by chemiluminescence with DIOGENES (National Diagnostics, Atlanta, GA), a superoxide-specific, luminol-based detection system, according to the manufacturer's instructions. Measurements were performed in 96- well microtiter chemiluminescence plates (5x10 4 cells per well). The total integrated light units recorded (Spectramax M5, Molecular Devices, Sunnyvale, CA) from siRNA NOX4 transfected cells were compared to those recorded in cells transfected with negative- control siRNA.
  • the present example demonstrates the location specificity of the dominant pathway to relapse in colon cancer. Attention is focused on samples in GSE14333 with Dukes stage A, B or C. Table 3 demonstrates the characteristics of patients in GSE14333. Table 3 Characteristics of patients in GSE14333
  • WNT5A wingless-type MMTV integration site family
  • member 5 A which is down regulated in the samples that will relapse.
  • Secreted frizzled-related protein 2 (SFRP2) which competes with the Wnt proteins for the Frizzled receptor, is up regulated.
  • the frizzled receptor, frizzled homolog 3 (FZD3) is down regulated in the relapse cases.
  • SFRP2 secreted frizzled-related protein 2
  • FZD3 frizzled homolog 3
  • Axin2 is down regulated in the relapse cases on the right side, reducing transcriptional inhibition by ⁇ -catenin.
  • Example 4 Single Genes are strongly predictive of Relapse in left-side and right- side tumors and encapsulate pathway activity
  • the multistate methodology is applied separately to the left-side tumors and the right-side tumors to identify multistate probes that are significantly predictive of relapse.
  • NOX4 219773_at
  • the distribution of NOX4 in the left-side tumors shows a large component with low mean expression and narrow variance, and a right tail of elevated expression ( Figure 1(a)).
  • the multistate methodology divides the two components at the expression value 3.0.
  • NOX4 expression is summarized as a binary variable by assigning 1 to every sample with expression level above the cut value (the NOX4+ component), and 0 for the other samples (NOX4-).
  • a CPH with a binary variable representing the MMP3+/- components has a -value 3.86 x 10 "6 .
  • NOX4 and MMP3 provide independent information about relapse since the poor prognosis components defined by the two genes have few cases in common.
  • the high component of family with sequence similarity 69, member A is enriched with relapse cases not identified by CDX2.
  • the multistate genes capture the pathogenic effects of the genes listed in Table 4 and Table 5 hence, the pathways containing these genes.
  • the most significant gene in the left-side analysis is integrin-binding sialoprotein (IBSP).
  • IBSP integrin-binding sialoprotein
  • the NOX4+ component in addition to containing the samples with elevated NOX4 expression contain the samples with elevated IBSP expression. Assessing the relationship quantitatively, a t-test for the mean expression value of IBSP in NOX4+ versus NOX4- has a -value of 1.38 x 10 "5 .
  • a CPH using IBSP expression as the variable, restricted to NOX4-, is not statistically significant, since the NOX4- component contains almost no samples with elevated expression of IBSP. In this way, IBSP can be replaced by NOX4 in a survival model.
  • the multistate gene MMP3 similarly represents the next most significant gene, WNT5A. In Table 4 and Table 5, for each probe, the multistate gene is identified that separates the gene's expression into high and low components in a statistically significant manner. On the right side, CDX2 effectively represents almost all of the probes listed in Table 5.
  • Tests that monitor only NOX4 would not be capable of distinguishing LCC from RCC disease.
  • the ability to distinguish LCC from RCC disease is possible here because of measuring expression levels of CDX2.
  • the inclusion of additional probes for CDX2 and/or FAM69A, as identified in Table 5, provides a much more robust analysis, and corrects an otherwise incorrect diagnosis of a colon cancer patient as at "low risk” for recurrent colon cancer relapse.
  • patients with RCC do not have low expression levels of NOX4, (below normal, non-cancerous tumor tissue), such patient tissue samples would be erroneously identified as "good prognosis" patients, with a good indication of colon cancer free survival.
  • the present example demonstrates the utility of the present invention for treating left-side colon cancer disease through targeted reduction of colon cancer cell superoxide production. Because elevated NOX4 is identified as being prognostic of a high probability of colon cancer disease relapse in left colon cancer disease, it is proposed that this model may be used to identify and screen for pharmaceutical agents useful in the improved treatment of patients identified to have left-side colon cancer disease.
  • ROS reactive oxidative species
  • Overproduction of reactive oxidative species (ROS) has long been recognized as a risk factor in carcinogenesis.
  • ROS reactive oxidative species
  • SW620 cells are a lymph-node metastasis colon adenocarcinoma cell line. NOX4 is shown to be expressed in this cell line, and the present example demonstrates that application of NOX4 siRNA causes a significant reduction in ROS production.
  • NOX4 inhibition with RNA interference in SW620 cells was found to be associated with a decrease in superoxide producing activities of the cells as indicated by the reduced ROS production ( Figure 5).
  • NOX4 function in metastatic SW620 cells the affect of NOX4 on cell viability was examined. NOX4 expression was silenced using RNAi interference by transfecting SW620 cells with oligonucleotides targeting the NOX4 transcript. Similar cell viability was observed between NOX4 silenced cells and negative control cells as reported in Figure 4. Therefore, targeted NOX4 knockdown does not seem to affect cell viability.
  • the microarray dataset GSE14333 analyzed here demonstrates that disease progression in RCC is dominated by elevated Wnt signaling and elevated proliferation, most strongly indicated by elevated levels of CCND1 in the relapse cases. Up regulation of CCND1 is accompanied by increased expression of the pro-apoptotic gene GADD45A and elevation of the growth arrest gene CDKN2B. Thus, these tumors that have not yet metastasized may be in a cycle of rapid mitosis and apoptosis.
  • the GSE14333 dataset is different from other datasets, such as GSE12945, GSE17536, GSE17537.
  • the cohort GSE14333 contains the patients in GSE17536, GSE17537, but it also contains samples not studied earlier.
  • NOX4 inhibition in SW620 shows no decrease in cell viability.
  • siRNA- mediation corresponds to a significant reduction in ROS production in the SW620 cells. This finding suggests that NOX4 is a novel source of ROS production in metastatic and pre-metastatic colon cancer.
  • NOX4 exerts cancer-promoting effects, it is most likely at more advanced tumor stages, as NOX4 expression is comparable to normal colon levels in primary adenocarcinoma and carcinoma derived cell lines and above normal colon levels in the metastatic cell line, SW620 ( Figure 3).
  • NOX4 is critical for HIF2-alpha transcriptional activity 20 . Specifically, inhibition of NOX4 decreases HIF2-alpha production. In the leftside colon cancer samples microarray data, there was no change in HIF2-alpha expression between the NOX4- and NOX4+ component. A change in hypoxia-related gene expression that was identified was a small decrease in HIGl hypoxia inducible domain family, member 1A and HIGl hypoxia inducible domain family, member 2A expression in NOX4+ over NOX4-. The results in Table 4 show that NOX4 expression is central to the progression of LCC.
  • MMP3 is a member of the matrix metalloproteinases family of extracellular proteinases that mediate many of the changes in the tumor microenvironment during cancer progression .
  • the genes correlated to MMP3 in expression point to a significant role for reduced Wnt signaling in left-side disease progression.
  • WNT5A is a known tumor-suppressor whose promoter is frequently methylated in colorectal cancer . In contrast, Wnt signaling is apparently elevated in the right-side colon cancer relapse cases.
  • CDX2 acts as a transcription factor, initially expressed during embryogenesis in the development of the small intestine and colon, and regulating a diverse range of functions from proliferation, cell-cycle arrest, differentiation, and apoptosis 22 .
  • CDX2 is expressed throughout the colon and regulated post-translationally through phosphorylation.
  • carcinogenesis expression patterns of CDX2 are altered. Analysis of 65 colorectal tumors mapping CDX2 expression throughout the colon and rectum found significantly lower expression of CDX2 in 37 right-sided, poorly differentiated tumors as compared to 28 left-sided tumors .
  • CDX2 Methylation of the CDX2 promoter has been proposed as a mechanism for down-regulation in colorectal carcinomas 24 .
  • CDX2 inhibits the Wnt signaling pathway, through reduction of the tyrosine phosphorylation of ⁇ -Catenin, resulting in decreased T-cell factor signaling and cell proliferation 27 .
  • CDX2 regulates E-cadherin trafficking to the cell membrane .
  • FAM69A is located at lp22.1, a genomic region that is preferentially deleted in microsatellite stable colon tumors 25 .
  • 9Q contains single nucleotide polymorphisms that increase the risk of multiple sclerosis . Expression patterns of the genes in this region do not show signs of deletion in the microarray data used here. The mechanism by which FAM69A expression is correlated with relapse risk remains an open problem for future study. [0156] It has previously been proposed that the differences in survival between RCC and LCC could be the results of any number of causes, for example difference in time of detection, embryologic origin, exposure to fecal matter or genetics 3 . Regardless of the underlying cause, different mechanisms dominate progression of RCC and LCC, establishing that they should be treated as different diseases. The prominent role of NOX4 as a prognostic biomarker in LCC makes it a important target for this cancer biology and LCC specific therapeutics.
  • Example 7 Genomic test for Separating Right-side colon cancer (RCC) from Leftside Colon Cancer (LCC) with a high degree of accuracy
  • Expression levels of the gene prostate cancer susceptibility candidate can be used to accurately estimate the location of origin of a colon tissue sample.
  • PRAC prostate cancer susceptibility candidate
  • a positive expression level of a gene is defined as having a detectable expression level by quantitative RT-PCR.
  • a negligible expression level of a gene, such as PRAC is defined as not having a detectable level of expression by quantitative RT-PCR.
  • a colon tumor sample that positively expresses the gene PRAC is very likely to be a left-side sample.
  • PRAC is very likely to be a right-side sample.
  • the prognostic tests for LCC and RCC disclosed in this invention uses one or more gene in its several embodiments, however, no increased prognostic power is found with more than two genes.
  • the efficient method for measuring the expression levels of few genes is quantitative RT-PCR.
  • RT-PCR quantitative RT-PCR.
  • one version of the test that could be used in a clinical setting will use RT-PCR to measure several species in mRNA from an FFPE tissue source. Because some mRNA species may be degraded in FFPE tissue, alternative tests will be sought using probes found in Table 1 and Table 2. This development process proceeds through the following two steps, separately for LCC and RCC.
  • RT-PCR probes that yield equivalent measurement of the mRNA species in frozen and FFPE preparations of the same colon tissue.
  • the identification of an RT-PCR probe is known to one skilled in the art of molecular biology.
  • the RT-PCR probe is an oligonucleotide of 15-60 nucleotides that hybridize with high specificity to the targeted species of mRNA.
  • FFPE colon cancer samples (RCC and LCC respectively) with known 5-year relapse status, select as the first gene in the panel the one that is most significantly prognostic.
  • the invention provides a computer implemented method of determining relapse free survival probability for a LLC or RCC patient having undergone colon cancer surgery.
  • the computerized method comprises classifying the colon cancer patient as a right side colon cancer (RCC) or as a left side colon cancer (LCC) disease patient by identifying the side of the colon on which the colon cancer was localized and providing said identifying classification to a receiver module, where the identifying classification of the patient is LCC disease, measuring an expression level of an RNA transcript or expression product of NOX4 in a colon cancer tissue obtained from the LCC patient, to provide a test NOX4 test level, and where the identifying classification of the patient is RCC disease, measuring an expression level of an RNA transcript or expression product of CDX2 in a colon cancer tissue obtained from the RCC patient, to provide a test CDX2 level, and providing said expression level data to a receiver module; and determining the relapse free survival probability of the LCC patient as good in a LCC patient tissue with a low NO
  • the method may further include a computer implemented step wherein the module is directed to generate a prognosis report of said LCC patient or RCC patient.
  • the invention provides a computer implemented method of determining the probability that a LCC or RCC patient will not be responsive to chemotherapy. In patients determined to have a low probability of being responsive to chemotherapy, the patient may be excused from chemotherapy after having undergone colon cancer surgery.
  • the computer implemented method of determining a probability of a lack of responsiveness to chemotherapy treatment in a patient having had surgical intervention for right side colon cancer (RCC) or left side colon cancer (LCC) comprises classifying the colon cancer patient as a right side colon cancer (RCC) or as a left side colon cancer (LCC) disease patient by identifying the side of the colon on which the colon cancer was localized and providing said identifying classification to a receiver module, where the classification of the patient is LCC disease, measuring an expression level of an RNA transcript or expression product of NOX4 in a colon cancer tissue obtained from the LCC patient, to provide a test NOX4 test level, and where the identifying classification of the patient is RCC disease, measuring an expression level of an RNA transcript or expression product of CDX2 in a colon cancer tissue obtained from the RCC patient, to provide a test CDX2 level, and providing said expression level data to a receiver module; and determining the likelihood of response to chemotherapy of the LCC patient as low in a patient with a low NO
  • an expression level is considered low or high as compared to a threshold value, wherein said threshold value is calculated from a reference set of like-gene expression levels from a like-classified colon cancer patient population, said like-classified patient population comprising relapse and relapse-free colon cancer patients not having received chemotherapy.
  • the method may further include a computer implemented step wherein the module is directed to generate a prognosis report of said LCC patient or RCC patient.
  • probes that have an oligonucleotide length of at least about 20 to 70 nucleotides and that have binding affinity for the biomarker genes identified here (NOX4, CDX2, MMP3, FAM69A) may be identified and used according to the present invention employing the teachings rendered here together without an undue amount of trial and error.
  • Standard molecular biology techniques and teachings such as those provided in Carlson, S., et al. (2011), Molecular Biology Techniques, 3 rd Edition, Academic Press, may be used to identify specific oligonucleotide probes, and then used together with or instead of those specific genetic probes identified here with equal if not improved efficacy.

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Abstract

La présente invention concerne le fait que le risque de rechute dans le cas de tumeurs du côlon droit se caractérise par une progression du cycle cellulaire accéléré et une signalisation Wnt élevée, et le risque de rechute dans le cas de tumeurs du côlon gauche se caractérise par une signalisation Wnt réduite. L'indicateur du biomarqueur de pronostic de gène unique pour le cancer du côlon gauche est le NADPH oxydase 4 (NOX4), le MMP3, ou un ensemble des deux gènes. Un indicateur du biomarqueur de pronostic génétique pour le cancer du côlon droit est l'homéoboîte de type caudal 2 (CDX2), le FAM69A, ou un ensemble des deux gènes. Les niveaux d'expression de NOX4 chez des patients humains atteints d'un cancer du côlon gauche sont utilisés dans des stratégies de traitement clinique propre au patient. Des patients atteints d'un cancer du côlon gauche dans lequel la tumeur présente un faible niveau d'expression de NOX4 ont une probabilité élevée de survie sans rechute à 5 ans, tandis qu'un niveau élevé d'expression de NOX4 indique une plus faible probabilité de survie. Les niveaux d'expression de CDX2 chez les patients humains atteints d'un cancer du côlon droit sont utilisés dans des stratégies de traitement clinique propre au patient. Dans des cancers du côlon droit, un niveau élevé d'expression de CDX2 indique une probabilité élevée de survie sans rechute à 5 ans, tandis que des niveaux d'expression plus bas indiquent une probabilité inférieure de survie sans rechute à 5 ans. Le NOX4 (CDX2) permet un meilleur pronostic pour les traitements du cancer du côlon gauche (côlon droit). Le NOX4 est exprimé dans un adénocarcinome. L'ARNsi de NOX4 entraîne une baisse significative de la production d'espèces réactives oxydantes.
PCT/US2011/066233 2010-12-20 2011-12-20 Biomarqueurs et leurs utilisations dans le pronostic et les stratégies de traitement du cancer du côlon droit et du cancer du côlon gauche WO2012088146A2 (fr)

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WO2015000906A1 (fr) * 2013-07-01 2015-01-08 Centre National De La Recherche Scientifique (C.N.R.S) Procédé de détection, d'identification et/ou de quantification d'un pathogène chez un individu
CN114540499A (zh) * 2022-03-17 2022-05-27 郑州源创吉因实业有限公司 基于pcd相关基因组合构建的模型在制备预测结肠腺癌预后产品中的应用

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US20030186302A1 (en) * 2002-03-29 2003-10-02 Yixin Wang Colorectal cancer diagnostics
US20050014165A1 (en) * 2003-07-18 2005-01-20 California Pacific Medical Center Biomarker panel for colorectal cancer
FR2919061B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de la plastine-i pour le diagnostic in vitro du cancer colorectal.
AU2010248803B2 (en) * 2009-05-14 2014-05-29 Arizona Board Of Regents On Behalf Of University Of Arizona Carcinoma diagnosis and treatments, based on ODC1 genotype

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CARLSON, S. ET AL.: "Molecular Biology Techniques", 2011, ACADEMIC PRESS
See also references of EP2655663A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015000906A1 (fr) * 2013-07-01 2015-01-08 Centre National De La Recherche Scientifique (C.N.R.S) Procédé de détection, d'identification et/ou de quantification d'un pathogène chez un individu
CN114540499A (zh) * 2022-03-17 2022-05-27 郑州源创吉因实业有限公司 基于pcd相关基因组合构建的模型在制备预测结肠腺癌预后产品中的应用

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